FTY720 but Not Its Immunosuppressive Phosphorylated Form FTY720-P Exerts Anti-Leukemic Activity towards Ph(+) and Ph(−) Myeloproliferative Disorders through Reactivation of the PP2A Tumor Suppressor.

Abstract 3259

Poster Board III-1

We have shown that the sphingosine analogue FTY720 markedly induces apoptosis of Ph(+) CML (chronic phase and blast crisis) and B-ALL progenitors regardless of their sensitivity to tyrosine kinase inhibitors through the reactivation of the tumor suppressor PP2A (protein phosphatase 2A). Here we report the identification of the molecular mechanism underlying the ability of FTY720 to activate PP2A, and its therapeutic potential towards Ph(+) and Ph(−) myeloproliferative disorders (MPDs).

First, we show that the pro-apoptotic and anti-proliferative effect of FTY720 is not limited to BCR/ABL⁺ leukemias but can be extended to Jak2^V617F-driven MPDs that are also characterized by the extensive (80% inhibition) Jak2^V617F-mediated suppression of PP2A activity. In fact, shRNA-mediated interference with the hnRNP A1-SET inhibitory pathway in Jak2^V617F-expressing erythro-myeloid precursors revealed that Jak2^V617F, like BCR/ABL, utilizes these effectors to impair PP2A function in a dose- and kinase-dependent manner. FTY720 treatment (0.5-1mM) fully restored PP2A activity and resulted in decreased BFU-E colony formation of Jak2^V617F-transduced Lin^- mouse marrow progenitors. Likewise, FTY720 decreased Jak2^V617F expression/activity and inhibited clonogenicity of Jak2^V617F-expressing 32D-EpoR+, Ba/F3 and TF-1 cells in PP2A/SHP-1-dependent manner.

Secondly, we show that PP2A is also inhibited in a SET-dependent manner in CD34⁺/CD38^- stem cells (HSCs) from CML patients. In Ph(+) HSCs, FTY720 but not imatinib or dasatinib decreases the number of long-term culture initiating cells (LTC-IC) and quiescent stem cells (CFSEmax) through activation of BCR/ABL-independent caspase-mediated apoptosis. Importantly, enhanced self-renewal of Ph(+) HSCs seems to depend on constitutive nuclear β-catenin transcriptional activity. In fact, FTY720 or PP2Ac lentiviral transduction, but not imatinib/dasatinib, induces b-catenin inactivation/degradation as measured by in situ immunofluorescence and LET/TCF assays. The effect of FTY720 in Ph(+) HSCs relies on the PP2A ability to override the BCR/ABL-independent inactivation of the β-catenin negative regulator GSK-3β, as treatment with the GSK-3β inhibitors LiCl and SB216763 increased the CFSEmax fraction and hampered the pro-apoptotic effect of FTY720 on quiescent Ph(+) HSCs. Notably, FTY720 did not affect normal stem/progenitor cell viability. Similar results were obtained with the LSK fraction from SCL-tTA/BCR/ABL mice.

Reportedly, FTY720 to act as an immunosuppressant requires phosphorylation by sphingosine kinase (SPHK) 2. To determine whether conversion of FTY720 into FTY720-P is important for its anti-leukemic activity in BCR/ABL- and Jak2^V617F-expressing cells, we used growth factor-dependent hematopoietic cells transformed by the constitutive activity of these oncogenic tyrosine kinases. In Jak2^V617F- and BCR/ABL-transformed cells, PP2A activation by FTY720 (2.5μM, 6h) is not changed when phosphorylation is prevented by treatment with the SPHK inhibitor dimethylsphingosine (2.5μM, 6h). Accordingly, FTY720-P did not activate PP2A and did not affect BCR/ABL- and Jak2^V617F-driven colony formation, indicating that the anti-leukemic activity of FTY720 does not require its phosphorylation. Because we have shown that sphingolipid PP2A activator ceramide specifically binds to SET, thus disrupting the SET/PP2A interaction in non hematopoietic cells, we investigated the effect of BCR/ABL or Jak2 V617F and that of FTY720 on ceramide production. LS-MS mass-spectrometry showed that levels of ceramide were similar in untreated and imatinib-treated BCR/ABL⁺ cells. Similarly, levels of diacylglycerol (DAG) kinase-phosphorylated ceramide were unchanged by treatment with FTY720, suggesting that oncogenic tyrosine kinase-induced suppression of PP2A and FTY720-dependent PP2A reactivation are not ceramide-mediated. Accordingly, FTY720 displaces biotin-labeled C6-ceramide (10μM) from SET, as measured by ceramide affinity chromatography followed by determination of SET levels in the eluted fraction.

Thus, FTY720 represents a powerful therapeutic tool as it has the potential to treat and, perhaps, eradicate Ph(+) and Ph(−) MPDs by efficiently inhibiting oncogene-dependent and -;independent signals through the reactivation of the tumor suppressor PP2A via disruption of the SET/PP2A inhibitory complex.